Insulin-like growth factor-I (IGF-I) promotes normal and adaptive growth of the small intestine and colon. Despite these beneficial actions, IGF-I is implicated as a mediator of intestinal tumorigenesis and is a potent inhibitor of crypt apoptosis. This proposal aims to examine intrinsic mechanisms which may limit the tumorigenic potential of IGF-I in the normal intestine while favoring normal growth and differentiation. Specifically we will test the hypothesis that low levels of IRS-1 favor increased apoptosis and high levels of SOSC2 favor normal growth and differentiation. Studies will use a model of irradiation induced apoptosis and cell cycle arrest, in vitro stem cell assays, model intestinal epithelial cell lines and mice with targeted deletion of key genes implicated in regulating IGF-I action. Aim 1 will test the hypothesis that partial or absolute IRS-1 deficiency impairs survival of normal or genetically damaged crypt stem cells and limits the anti-apoptotic actions of IGF-I in vivo and in vitro. Crypt apoptosis will be examined in non-irradiated and irradiated mice with 2,1, or 0 copies of the IRS-1 gene, and IRS-1 null and WT given vehicle or IGF-I. Clonogenic assays on isolated crypt epithelial cells from mice with 2.1 or 0 copies of IRS-1 will test directly whether there is a dose dependent requirement for IRS-1 as a mediator of survival or clonal growth of crypt stem cells or IGF-I action on stem cell survival. Known mediators of IRS-1/IGF-I action in cell lines will be quantified by assays on tissue extracts and isolated crypts and by immunohistochemistry. This will provide novel information about the in vivo mediators of IGF-I/IRS-1 action on crypt epithelial cells and define if IRS-1/IGF-I dependent changes in mediators occur selectively or preferentially in crypt stem cells. Aims 2 and 3 will test the hypothesis that suppressor of cytokine signaling-2 (SOCS2) limits the proliferative actions of IGF-I on intestinal epithelial cells and promotes enterocyte differentiation. Caco2 cells, HT-29 cells and IEC-6 cells transfected with SOCS2 antisense or sense expression constructs will be used to define the mechanisms by which SOCS2 reduces IGF-I dependent proliferation, promotes differentiated function and to assess if SOCS2 affects apoptosis. Mice with targeted disruption of one or both SOCS2 alleles and wild type littermates will be used to define the in vivo role of SOCS2 in post-natal growth and maturation of the intestine and test directly if SOCS2 limits the proliferative or anti-apoptotic actions of IGF-I in intestine in vivo.